Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis

Brian P. Weiser, James T. Stivers, Philip A. Cole

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that prevents the stable incorporation of deoxyuridine monophosphate into DNA in the form of U/A basepairs. During S-phase, UNG2 remains associated with the replication fork through its interactions with two proteins, Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which are critical for DNA replication and repair. In this work, we used protein semisynthesis and fluorescence anisotropy assays to explore the interactions of UNG2 with PCNA and RPA and to determine the effects of two UNG2 phosphorylation sites (Thr6 and Tyr8) located within its PCNA-interacting motif (PIP-box). In binding assays, we found that phosphorylation of Thr6 or Tyr8 on UNG2 can impede PCNA binding without affecting UNG2 catalytic activity or its RPA interaction. Our data also suggests that unmodified UNG2, PCNA, and RPA can form a ternary protein complex. We propose that the UNG2 N-terminus may serve as a flexible scaffold to tether PCNA and RPA at the replication fork, and that post-translational modifications on the UNG2 N-terminus disrupt formation of the PCNA-UNG2-RPA protein complex.

Original languageEnglish (US)
Pages (from-to)393-401
Number of pages9
JournalBiophysical Journal
Issue number2
StatePublished - Jul 25 2017
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics


Dive into the research topics of 'Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis'. Together they form a unique fingerprint.

Cite this